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- The following stock solutions are available to make a protein extraction buffer: 100% Nonidet P-40, 1 M Tris-Cl, and 0.5 M EDTA. What quantity of the original stocks will be needed to make 250 ml of buffer with the following final concentrations: 0.5% Nonidet, 150 mM Tris-Cl, and 10 mM EDTA?The Kjeldahl method is commonly applied to food samples for the determination of total protein content. (a.) Outline the procedures of Kjeldahl method. (b.) Calculate the per cent protein of the food sample if the following results are obtained: -mass of food samples: 1.015 g -Normality of HCL: 0.1 N - Volume of HCL used for sample: 22.0 mL - Volume of HCL used for blank: 0.2 mL - Conversion factor of per cent N in protein: 6.25 (c.) The value of total protein content obtained from (b.) was much higher than that expected. Suggest one possible reason and the corresponding solution for improvement of the analysis.An ion-exchange chromatographic separation is performed using a diethyl-aminoethyl- (DEAE)-sepharose column to separate proteins in a mixture. The mixture contains Protein A (pl=6.0), B (pl35.0), C (pl=7.5), D (pl =1), and E (pl=4.0). The protein mixture is prepared in a buffer solution pH =5. When the protein mixture is loaded onto the column, and the column is washed with a buffer solution pH 5, which protein(s) will be captured by DEAE-sepharose in the column? O Protein B because it is predominantly in net negative charge form. O Proteins D and E because they have predominately net negative charge in pH 5 solution O Proteins A, C, D and E because they have charges O Proteins A and C because both both predominantly have net positive charges O Proteins Band E because both predominantly have net positive charges
- 1. Lactoferrin is an iron-binding glycoprotein that forms the antibiotic fragment lactoferricin during digestion. The protein was isolated from 1.5 liters of ewe skim milk by successive ammonium sulfate precipitation until a final salt concentration of 70% was obtained. Determine the amount (in g) of powdered ammonium sulfate needed in bringing the solution from (a) 0% to 40% saturation and (b) 40% to 70% saturation assuming that the volume remains constant. Initial concentration of (NH4)2SO4, % saturation 0 10 20 30 40 50 60 70 80 90 10 55 Final concentration of (NH4)2SO4, % saturation 30 40 50 60 70 80 Grams of solid (NH4)2SO4 to be added to 1 L of solution 176 242 314 390 472 561 657 117 405 491 584 59 421 351 438 20 113 57 182 121 61 251 325 63 188 126 195 130 65 260 337 270 202 281 135 67 210 140 70 90 761 685 511 609 533 365 292 219 146 HUGH LATLE 100 73 457 381 305 228 152 76(a) The octapeptide AVGWRVKS was digested with the enzyme trypsin . Which method would be most appropriate for separating the products: ion-exchange or gelfiltration chromatography? Explain. (b) Suppose that the peptide was digested with chymotrypsin . What would be the optimal separation technique? Explain.1) You have been asked to make up four 1.5% agarose gels at 30 ml each. Ethidium Bromide is to be added at 0.5 ug/ml and you have a bottle at 10ug/ml. Write down step-by-step protocol of how to proceed. 2. How would you make up one liter of 1X TAE buffer using a 25X stock?
- The protein concentration of a known standard is 100mg/mL If you prepared a serial dilution, mixing 10μL of protein with 40μL of water what would be concentrations of the first 3 dilutions?Given the following information, calculate the total activity in the undiluted protein sample. Activity of 1 ml of diluted sample = 0.5 Total volume of sample = 5 ml Dilution factor = 10 25 50.5 250 2.5Answer the following questions about protein concentration determination. a) At what wavelength should an albumin protein solution placed in a quartz cuvette be measured in the spectrophotometer? Why is that? b) With which methods other than spectrophotometer can the amount of protein be measured quantitatively?
- 1) Calculate the required gram of Tris-Hcl to prepare 1M, 100 ml Tris-HCl (Tris Stock) with mol and Molarity formula. 2)Calculate the required gram of EDTA to prepare O.5M, 100 ml EDTA (EDTA stock) mol and Molarity formula. 3.) Calculate the required volume of Tris Stock to prepare buffer A which contains 25 mM Tris-Hcl with final volume is 100 ml by using M1V1 =M2V2 formula 4) Calculate the required volume of EDTA stock to prepare buffer A which contains 10 mM EDTA with final volume is 100 ml by using M1V1 =M2V2 formula 5)Do the same for Buffer B calculationsHOW MANY of the following five items represent modes of protein denaturation? 1.) heating a protein sample 2.) cooling a protein sample (slowly) 3.) putting a protein sample in a saltier environment 4.) eating a protein sample (such as with a whisk) 5.) adding acid to a protein sampleWhat is the basis for the separation of proteins by the following techniques? (a) gel-filtration chromatography(b) affinity chromatography(c) ion-exchange chromatography